Reinnervation Patterns of Hindlimb Muscle Neuromuscular Junctions after Nerve Injury
Katherine Bernadette Santosa, M.D.1; Bianca Vannucci, BA1; Alexandra Marie Keane, B.A.1; Katherine G Campbell, B.S. candidate1; Albina Jablonka-Shariff, Ph.D.2; Alison K. Snyder-Warwick, MD3; (1)Washington University, Saint Louis, MO, (2)Division of Plastic and Reconstructive Surgery, Washington University, Saint Louis, MO, (3)Plastic Surgery, Washington University School of Medicine, St. Louis, MO
Purpose: Several outcome measures to assess nerve regeneration and muscle reinnervation following nerve injury have been described in the literature. Many of these techniques, however, are fraught with limitations such as low sensitivity, need for specialized equipment or mouse lines, and evaluation in an ex vivo environment. Here, we present a technique to directly assess motor recovery via confocal microscopic imaging of motor endplate reinnervation. The purpose of our study is to evaluate all neuromuscular junction (NMJ) structures (axon terminals, Schwann cells (SCs), and motor endplates) during hindlimb muscle reinnervation following sciatic nerve transection and repair.
Methods: Adult wildtype (C57BL/6) mice underwent transection of the right sciatic nerve with immediate repair and were randomized to groups corresponding to the time of evaluation (t=0, 1, 2, 3, 4, 8 or 30 weeks) after nerve injury. At the time of harvest, the extensor hallucis longus (EHL) and extensor digitorum longus (EDL) muscles, both innervated by the deep peroneal branch of the sciatic nerve, were collected for whole mount confocal microscopy. NMJ morphology was characterized and reinnervation quantified by imaging axons (NF200), SCs (S100), and motor endplates (?-bungarotoxin).
Results: At one week after sciatic nerve transection and repair, all endplates were denervated. At two weeks, ~54% of endplates were partially innervated. At 3 weeks, nearly 90% of all endplate were at least partially innervated, and NMJs showed evidence of sprouting and polyaxonal reinnervation. At 8 weeks, all endplates were reinnervated, although evidence of sprouting axonal connections between endplates remained. At 30 weeks following injury, single innervation of the NMJ was restored but endplate fragmentation, not seen at earlier time points after nerve injury, was evident.
Conclusions: The abundance of NMJs and the thin muscle bulk of the EHL and EDL make ideal models for evaluation of motor reinnervation following nerve injury. Reinnervation following murine sciatic nerve transection and repair occurs rapidly and is completed by ~3 weeks after injury; however, the normal 1:1 axonal to endplate relationship is not fully restored by 2 months after nerve injury. In the absence of transgenic mouse models whose axons and/or SCs express fluorescent chromophores, we present a consistent and feasible method for evaluating motor reinnervation following nerve injury.
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